Project Summary Latently infected CD4+ T cells represent the major barrier to HIV-1 eradication. Many strategies are being developed to eliminate these cells, but the field lacks a simple sensitive assay that can screen for these cells. PCR based assays cannot distinguish between replication-competent versus defective virus and recent studies have shown that the current state of the art culture assay measures only a small fraction of the replication-competent virus present. In this proposal we aim to develop a sensitive assay that is capable of selectively amplifying replication-competent virus from latently infected CD4+ T cells. Such an assay could inform the decision of whether or not to discontinue cART in patients after a therapeutic intervention is made. The need for such an assay is highlighted by the recent report of the 2 ?Boston patients? who were treated with allogeneic stem cell transplantation for malignancies. No HIV DNA was detected by PCR in either patient from as many as 200 million PBMCs (<0.07 copies/106 PBMCs), but after cART was discontinued there was a rapid rebound in viremia in both patients. This outcome is undesirable is it negates the theoretical advantages to having smaller HIV-1 reservoirs. The development of a simple but sensitive assay that can screen a very large number of CD4+ T cells may help prevent the occurrence of a similar scenario from occurring in the future. We will use the optimized assay we develop to screen CD4+ T cells from patients who were treated with ART during primary infection and correlate the number of latently infected cells as measured in our assay to the time it took for viral rebound to occur when ART was discontinued in these subjects. This work will be important for the development of strategies for HIV-1 eradication since the assay will help inform the decision of whether or not to discontinue antiretroviral therapy.